Role of miR-944/MMP10/AXL- axis in lymph node metastasis in tongue cancer

Occult lymph-node metastasis is a crucial predictor of tongue cancer mortality, with an unmet need to understand the underlying mechanism. Our immunohistochemical and real-time PCR analysis of 208 tongue tumors show overexpression of Matrix Metalloproteinase, MMP10, in 86% of node-positive tongue tumors (n = 79; p < 0.00001). Additionally, global profiling for non-coding RNAs associated with node-positive tumors reveals that of the 11 significantly de-regulated miRNAs, miR-944 negatively regulates MMP10 by targeting its 3’-UTR. We demonstrate that proliferation, migration, and invasion of tongue cancer cells are suppressed by MMP10 knockdown or miR-944 overexpression. Further, we show that depletion of MMP10 prevents nodal metastases using an orthotopic tongue cancer mice model. In contrast, overexpression of MMP10 leads to opposite effects upregulating epithelial-mesenchymal-transition, mediated by a tyrosine kinase gene, AXL, to promote nodal and distant metastasis in vivo. Strikingly, AXL expression is essential and sufficient to mediate the functional consequence of MMP10 overexpression. Consistent with our findings, TCGA-HNSC data suggests overexpression of MMP10 or AXL positively correlates with poor survival of the patients. In conclusion, our results establish that the miR-944/MMP10/AXL- axis underlies lymph node metastases with potential therapeutic intervention and prediction of nodal metastases in tongue cancer patients.

8. Supplement SFigure3 is mentioned before SFigure2. Please check for the correct order. 9. Did the authors consider other methods to study proliferation as passaging and counting by hematocytometer does not accurately measure proliferation (could be mix of differences is viability, proliferation, apoptosis) and is prone to errors. Did the authors test differences in apoptosis separately?
10. Some methods properly describe 1) the number of individual experiments performed and 2) number of technical repeats in each experiment. However, some lack this information. Please add this information for all experiments. Without this information, it is impossible to evaluate the results.
11. There is some uncertainty (partly because of the above-mentioned shortages on repetitions of experiments) on how statistical analyses were performed in each experiment. For example, in Figure  2, many of the Figures show relative values to control (=1 or 100). From which values were the P values calculated? It would be more informative to illustrate the values used for statistical analyses in Figures and not the ratios to control. Also, for 2E, 2I, the statistics are confusing. Are the controls (values from technical repeats, individual experiments?) compared to all sh cells? The differences illustrated by these experiments seems clear, but statistics should be performed properly and described thoroughly. Without this information, it is impossible to evaluate the results.
12. Do the authors have reason to use P values <0.005 for ** and <0.0005 for *** as more commonly used values would be <0.01 and <0.001.
13. For Western blots, molecular weight standards should be marked (for all) figures and original, whole blot images should be provided as Supplementary data. Without this information, it is impossible to evaluate the results.
14. How many times the blots were performed? For at least most important differences claimed based on the results, three blots should be performed and the differences in band intensities measured and related to housekeeping protein.
15. In Figure 2, two separate blots for vinculin as housekeeping are shown. However, twelve proteins were detected and presumably, they are not all from two different blots with vinculin in addition. If this is the case, housekeeping should be provided for all blots separately. Without this information, it is impossible to evaluate the results.
16. IHC: the authors do not provide information for the antibody used? Should there be also isotype control to show the antibody specificity -that depends on the antibody used. Without this information, it is impossible to evaluate the results.
17. The authors should describe in the text (and for this, please provide higher magnification images along with the current ones) where is the MMP10 IHC staining localized -is it inside the cells, on membrane, extracellular? Also, the distribution of MMP10 in tumors would be highly informative and interesting -e.g. is it expressed in the invasive front, close to certain tissue structures etc.?
Reviewer #2 (Remarks to the Author): In this study, the authors examined the role of miR-944/MMP10/AXL-axis in node-positive tongue tumors. Authors found that MMP10 overexpression was frequently observed in node-positive tongue tumors and knockdown of MMP10 suppressed proliferation, migration, and invasion. Moreover, authors showed that miR-944 negatively regulates MMP10 by targeting its 3'-UTR. Among upregulated genes by MMP10 overexpression, authors focused on AXL and miR-944/MMP10/AXL-axis was involved in tumor progression both in vitro and in vivo. In conclusion, authors establish that the miR-944/MMP10/AXL-axis underlies lymph node metastases with potential therapeutic intervention and prediction of nodal metastases in early-stage tongue cancer patients.
It is well-known that MMP10 is involved in tumor progression. In this study, it is still unclear the detailed mechanism of miR-944/MMP10/AXL-axis in node-positive tongue tumors via promoting proliferation, migration, and invasion. I feel that this paper is too preliminary for publication.
My comments are as the following: 1. In Figure 1, authors showed that MMP10 overexpression was involved in tongue cancer patients with lymph node metastasis. However, authors previously reported that MMP10 overexpression was associated with lymph node metastasis in early tongue cancer patients. Even if the tumor size is small (T1 and T2), if patients have metastasis, authors cannot say early stage. Authors should not use the term "early stage". Moreover, authors should explain why you focused on T1 and T2 cancer. Moreover, in your previous study, authors used HPV negative early-stage tongue cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco. In general, cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco have buccal cancer. Why authors focused on tongue cancer in previous and current studies? 2. Authors should demonstrate that the involvement of miR-944/MMP10/AXL-axis is specific in T1 and T2 tongue cancer. To prove your hypothesis, authors should explain the following question. 1) How about the role of miR-944/MMP10/AXL-axis in T3 and T4 tumors? 2) How about the involvement of miR-944/MMP10/AXL-axis in early-stage cancer (T1-T3 without metastasis)? 3) How about the role of miR-944/MMP10/AXL-axis in the precancerous lesions (ex. epithelial dysplasia)? 4) How about the role of miR-944/MMP10/AXL-axis in HPV-positive cancers? 3. Authors should examine the MMP10 overexpression and check the upregulation of AXL. In addition, authors should check the phenotype of AXL overexpression. 4. Authors showed that MMP10 overexpression promotes proliferation, migration, and invasion via AXL overexpression. How about the mechanism (how AXL regulates proliferation, migration, and invasion)?
Reviewer #3 (Remarks to the Author): In the manuscript "miR-944/MMP10/AXL-axis Mediates Lymph Node Metastasis in Early-Stage Tongue Cancer" by Dharavath et al, the authors validate the overexpression of MMP10 in tongue tumor sample and correlated with nodal metastasis. They also performed functional analysis in vivo and demonstrated that MMP10 is essential for tumor growth and nodal metastasis in mouse model. The authors also studied the mechanistic effect of MMP10 upregulation and demonstrated that miR-944 negatively regulates MMP10, and AXL signaling pathway mediates phenotypes associated suppressing proliferation, invasion, and migration of target tongue cancer.
Although the study is comprehensive and the amount of data generated are valuable, some concerns should be clarified in order to improve the quality and the impact of the manuscript.

Major comments:
The main criticism is that some experiments, specially those focusing in the mechanism of MMP10 regulation in tongue cancer, lacks the appropriate control and some data are not consistent through the manuscript.
1-) The main finding in the paper relies on the proposed regulatory mechanism of miR-944/MMP10/Axl axis. In this regard, it is not clear why the authors did not evaluate the basal expression level of Axl in all cell lines employed in this study. For instance, only the levels of MMP10 were demonstrated ( Figure  S2). However, the authors did not show the levels of Axl in the cell. Does Axl levels correlate with MMP10 levels in all cell lines? And in tissue samples? 2-) What are the activation status (phosphorylated form) of Axl in the cell lines? And in the tissue samples?
3-) The mechanistic analysis was performed in MMP10 overexpressed cells. Considering that AW8507 and CAL27 express high levels of MMP10, why the authors overexpress this protein in a cell line and did not evaluate the mechanism in AW8507 and/or CAL27? 4-) Since the authors are claiming that MMP10 induces Axl expression, it would be important to demonstrate whether MMP10 indeed modulates Axl and proteins in the Axl pathway expression. In this regard, would be interesting to use MMP10 knocked down and evaluate Axl and Axl pathway protein levels 5-) There is lack of consistency in the western blots. MMP10 expression levels are completely different for cell line AW13516 in figure 3I and figure S2C (Levels of MMP10 in figure S2 is different when compared to Figure 3I). Why did the authors used both GAPDH (figure S2C) and vinculin (figure 3I) as loading control? 6-) Quantification and normalization of proteins in the Western blots protein should be performed. 7-) A figure summarizing the regulation of miR-944/MMP10/Axl axis in this model, should be included in the manuscript.
Reviewer #4 (Remarks to the Author): Review report for Dharavath et al. manuscript (Communications Biology) This study by Dharavath et al. describes the pro-metastatic functional effects of matrix metalloproteinase 10 (MMP10) on oral tongue cancer (OTSCC) both in vitro and in vivo. Furthermore, they demonstrate the regulation of MMP10 expression by miRNA-944 and discover the downstream signaling molecules (namely AXL), which transmit the functional effects of MMP10. Additionally, the use of MMP10 as a prognostic marker in OTSCC is explored. The researchers have used extensive molecular and cell biology methods as well as bioinformatic tools, which is the strength of this study and increases the quality of the research and reliability of the presented conclusions. The findings in this study are novel related its functional effects of MMP10 on OTSCC cells as well as regulation and signaling of MMP10 in OTSCC. Otherwise, the presented results support the previous findings on this topic. Previous research on this specific topic is limited in general, which also warrants the publication of this study together with its depth and validity. Yet, there are minor corrections to be made (suggestions given below) before a full approval can be given -these corrections should also improve the readability and impact of this manuscript even further.

Abstract:
The abstract is compact and nicely written, but some of the presented conclusions are possibly misleading. 1. Please consider revising the statement on the second sentence (rows 37-38). This result is not exactly clear in results section (see comment x) nor does the given n correspond to the number of node-positive tumors.
2. The sentence starting from row 41 "Next, using orthotopic tongue cancer model…" gives the impression that all functional aspects (proliferation, migration and invasion) were studied in the mouse model, which based on the results, is not the case (they were studied in vitro). Please consider revising this sentence. 3. For Keywords, please consider including AXL or miRNAs instead of "Epithelial mesenchymal transition" as they could better reflect the results and conclusions of this manuscript.

Introduction:
Introduction section gives the necessary overview on the research topic and question by discussing related literature. Yet this section could benefit from few changes as suggested below: 4. LNM is indeed found to result in worse prognosis for OTSCC patients, as suggested by authors in first part of the introduction (rows 60-62). Yet the references used do not discuss OTSCC but other cancers, although such research and associated literature exists. Please consider referring to OTSCCrelated publications or modifying the sentence accordingly. 5. Row 70: Please consider the term "lymphogenesis", should it read "lymphangiogenesis" instead? 6. The sentence on rows 74-76 only refers to ovarian cancer, although he referenced studies also include HNSCC and cervical cancer. Please consider revising this sentence. 7. Please consider toning down the conclusion in the sentence on rows 79-81. Even if MMP10 have been upregulated in another head and neck cancer and relates to pre-metastatic niche formation in cancers overall, the variation between cancers overall and within HNSCCs is great. Thus, it is hard to convincingly suggest that MMP10 would play a LNM-related role in OTSCC based on these factors, although it warrants the research on this topic.

Materials & Methods:
In general, the methods are clearly written out. Some clarifications could still be discussed. 8. On patient samples (Table I): Comment 8.1. This study focuses on tongue cancer, yet the patient cohorts also include tumors from the anatomical site of buccal mucosa. Could the authors explain why and describe, whether these tumors were excluded from the analyses or not? Comment 8.2. Could the authors describe what is the difference between smoking and tobacco status? Comment 8.3. What was the last follow-up date of these patients? Did it vary or were all followed to the suggested 5 years mark? 9. For western blotting (page 6 in M&M, page1-2 in supplementary M&M): How was the unspecific antibody binding blocked? 10. In general, the methods describe both technical replicates in assays as well as number of repeated experiments in a varying manner resulting this information to be missing in some assays (e.g. boyden chamber assays). Could the authors please provide this information for all the assays when necessary (this suggestion includes supplementary M&M).

Results:
This study provides a generous number of solid results to support the conclusions drawn in the next chapter. 11. For figure 1B: Could the authors explain why they have presented the gene expression values as negative delta Ct values? This is quite an uncommon presentation. 12. In the sentence on rows 251-254 authors claim OTSCC patients with LNM show higher gene and protein MMP10 expression, but seemingly the data is not provided for RT-PCR analysis. Please check. 13. For Table II: Authors claim significant results in text (rows 264-265), yet the table does not provide these. Please check and if needed, modify this sentence accordingly. 14. For figures 2 and 3: Please provide the molecular weights (or their markers) for the western blot images (this is also a recommendation by the journal). For further validation, whole blot images could also be provided for evaluation. 15. On page 16 authors describe the effect of high MMP10 or AXL expression on HNSC(C) patient survival. Have the authors checked if this conclusion can be made based on only OTSCC patients in this data? HNSCCs vary greatly in etiological and other factors (e.g. HPV), which might also affect survival. 16. On rows 379-381 authors claim that AXL could regulate metastasis, yet this conclusion is hard to justify only based on results from in vitro assays. Please consider toning down this conclusion. 17. On row 387 authors claim to have developed the orthotopic tongue cancer mouse model when indeed this model has been introduced by Myers JN et al already in their 2002 publication in Clin Cancer Res journal. Please consider using another verb in this sentence.

Conclusion:
18. Could the authors please clarify the comment on "elective neck dissection that is mandatorily performed" (juxtaposition, rows 416-419). Also please note that different countries might have different standards on elective neck dissection which should be taken into account. 19. Please check and possibly revise the use of term anti-correlated (unusual term, row 453) as well as consider if the sentences on rows 460-463 could be merged as they use the same references and discuss the same topic.
References: Please carefully check and correct the possible errors in references, see at least the following: 20. Reference 3 is apparently a book chapter. Please check the correct formatting for the reference type. We thank all the reviewers for their thorough analysis of our work. Please find the specifics of the changes made to the manuscript in blue, in response to the reviewers' recommendations.

Reviewer #1:
Comment #1: Please tone down the title as there is not enough in vivo evidence for such strong conclusion -although the role of miR-944 and AXL were examined in vitro, their role was not examined in mouse model to conclude them as mediators of LN metastasis. Response: As suggested by the reviewer, the title of the manuscript is changed to "Role of miR-944/MMP10/AXL-axis in Lymph Node Metastasis in Early-Stage Tongue Cancer". Comment #2: Abstract says: 'Next, using an orthotopic tongue cancer mouse model, we show that loss of MMP10 or miR-944 overexpression suppresses proliferation, migration, and invasion of tongue cancer cells impeding nodal metastasis.' This sentence should be changed as it gives an impression that all this was shown by mouse model. Instead, mouse model only provided data for the role of MMP10 and in vitro evidence (or partly human samples) supported the rest. Response: The abstract is revised and written as "...We demonstrate that proliferation, migration, and invasion of tongue cancer cells are suppressed by MMP10 knockdown or miR-944 overexpression. Next, we show that depletion of MMP10 prevents nodal metastases using an orthotopic tongue cancer mice model…". As an additional set of evidence to suggest sufficiency of MMP10 overexpression to promote metastases, we performed additional in vivo experiment of MMP10 overexpressing AW13516 cells injected orthotopically in the tongue of NOD-SCID mice and screened for metastasis. Interestingly, MMP10 overexpression is sufficient to promote nodal and distant metastasis in vivo. We have included the data in Figure 5, A-B and Supplementary Figure 13A in the revised manuscript (as shown below).

Figure 5 (A-B): MMP10 promotes metastases of tongue cancer in vivo. (A) IVIS imaging for detection of metastasis in mice injected (n=6/group) with AW13516 cells stably overexpressing empty vector or MMP10.
Bioluminescence imaging was performed after the resection of primary tongue tumors from the mice. The black arrowhead indicates the metastasis in regional lymph nodes and distant organs. (B) Tabular representation of the number of cells injected orthotopically into the tongue of mice and number of mice with metastasis in regional lymph nodes or distant organs.

Supplementary Figure S13 (A): IVIS imaging of mice organs showing metastasis in vivo. IVIS imaging of mice internal organs (sub-maxillary glands and cervical lymph nodes (M), lungs (L), liver (Li), kidney (K), spleen (S), uterus (U) and ovaries (O)) after necropsy to detect metastasis in mice injected with AW13516-vector control and MMP10-overexpression clones. Bioluminescence signal from the organs indicate metastasis.
The results are mentioned in the abstract as, "…overexpression of MMP10 leads to opposite effects upregulating epithelial-mesenchymal-transition, mediated by a tyrosine kinase gene, AXL, to promote nodal and distant metastasis in vivo..." Also, the experiment is detailed and merged with the earlier results of orthotopic tongue tumor study with MMP10 knockdown clones of AW8507 and CAL27 in the results section of the revised manuscript as, "…we established orthotopic tongue tumor mouse model using the MMP10 overexpression and knockdown clones. We injected luciferase-tagged AW13516 clones of MMP10 overexpression, and CAL27 and AW8507 clones of MMP10 knockdown (sh-2) along with vector control cells orthotopically into the tongue of 6 mice/group using needle gauge (30 G) after anesthetizing the mice with isoflurane. Caliper measurements at regular intervals showed that AW13516 cells form tumors after 25 days, CAL27 cells form tumors after 10 days, whereas, AW8507 cells formed tumors after 35 days of injection. However, the volume of AW8507-induced tumors was higher than CAL27-induced tumors, followed by AW13516-induced tumors. Interestingly, knockdown of MMP10 significantly reduced the tumor volume in both AW8507-induced and CAL27-induced compared to the control group (Supplementary Figure S12, A-B), suggesting the role of MMP10 in tumorigenesis of tongue cancer. Also, AW13516-and AW8507-induced tumors metastasized to cervical lymph nodes, lung, liver and kidney whereas, CAL27-induced tumors did not metastasize to any organ or lymph nodes (Supplementary Figure S12C, S13, A-C). Of note, the CAL27 cell line is reported to be a non-metastatic cell line in literature as well (Ji, J. et al. International journal of clinical and experimental pathology, 2018). In AW13516-induced tumors, metastasis was observed in 6/6 (100%) mice in the MMP10 overexpression group and 1/6 (16.7%) in vector control group. In AW8507-induced tumors, metastasis was observed in 6/6 (100%) mice in the control group and none (0/6) in the MMP10 knockdown group ( Figure 5, A-D), suggesting a significant increase in the metastasis of tongue cancer upon overexpression of MMP10 and decrease in metastasis upon depletion of MMP10…" Comment #3: Page 15. row 346: …'regulates lymph node metastasis.' please tone down as the conclusion is too strong based on the data. Response: Following the reviewer's suggestion, the sentence in the results section is now revised as "…suggesting that miR-944/MMP10 is potentially involved in regulating lymph node metastasis…".

Comment #4:
Conclusion that AXL is connected to miR-944 cannot be made solely based on the connection of MMP10 with miR-944 and MMP10 with AXL separately (abstract, results p15. row. 348 title) Response: We have modified the text in the abstract section as "…AXL expression is essential and sufficient to mediate the functional consequence of MMP10 overexpression…" Also, the title of the result section is revised to "…MMP10 driven invasion and migration of tongue cancer cells is mediated by the AXL signaling pathway…". Comment #5: Page 3. row 70: lymphogenesis to lymphangiogenesis. Please correct. Response: We apologise for the typo. We have now corrected the typographical error from "lymphogenesis" to "lymphangiogenesis". Comment #6: 'N-zero clinical trial', please provide short explanation for this. Response: An important prognostic factor for oral cancer is lymph node metastases. Due to occult lymph node metastases, which affects about 30% of patients with early-stage (T1 and T2) oral cancer, there are conflicting treatment recommendations. One method is elective neck dissection (END) which involve surgical excision of lymph nodes together with the original tumor, and the other is watchful waiting for patients who develop nodal metastases during follow-up therapeutic neck dissection (TND). On the basis of 500 patients with early-stage (T1 and T2) oral cancer, the N-zero clinical trial compared the survival advantages of END and TND. The study established, despite higher cost and higher morbidity associated, END as a standard of care for the early-stage clinically node negative (T1, T2 and N0) oral cancer patients. Based on reviewer's suggestion, we have included a short explanation on N-zero clinical trial in the results section of revised manuscript as "…On the basis of 500 patients with early-stage (T1 and T2) oral cancer, the N-zero clinical trial compared the survival advantages of END and TND. The study established END as a standard of care for patients with early-stage clinically nodal negative (T1, T2 and N0) oral cancer and showed its superiority in terms of overall and disease-free survival rates.…" Comment #7: Page 20, row 453. 'Anti-correlate' maybe other expression should be used? Response: The sentence is changed in revised manuscript as "…miR-944 expression is significantly negatively correlated with MMP10 expression…". Comment #8: Supplement SFigure3 is mentioned before SFigure2. Please check for the correct order. Response: We apologize for the error. The order of all the figures is now updated as per citation in the results section of the revised manuscript.
Comment #9: Did the authors consider other methods to study proliferation as passaging and counting by hematocytometer does not accurately measure proliferation (could be mix of differences is viability, proliferation, apoptosis) and is prone to errors. Did the authors test differences in apoptosis separately? Response: We thank the reviewer for this query. We have now performed additional experiments to address the reviewer's query. In brief, we performed cell viability and apoptosis experiments with MMP10 knockdown clones of AW8507 and CAL27 cell lines, along with a western analysis to check the levels of Caspase-3 and PARP cleavage. We were unable to conduct an apoptotic experiment using Annexin V-FITC labelling because the shRNAs of the MMP10 knockdown clones of AW8507 and CAL27 were GFP-tagged. As a result, we used AW8507 and CAL27 cells that had MMP10 knocked down using siRNA for the experiment. The data is now included in the manuscript as Supplementary Figure S3 (as shown below with the legend):

Supplementary Figure S3: Knockdown of MMP10 does not affect apoptosis of tongue cancer cells. (A and E) Bar plot of cell viability assay performed using Propidium Iodide (PI) staining indicates the percentage of late apoptotic and necrotic cells in MMP10 knockdown clones (sh-1, sh-2 and sh-3) and non-targeting shRNA control clones (sh-NT) of AW8507 (A) and CAL27 (E). (Bottom Western blots) Western blots indicate the expression of Caspase 3 and PARP cleavage in the MMP10 knockdown and vector control cells. β-actin or Tubulin was used as loading control. (B and F) qRT-PCR analysis indicating siRNA mediated knockdown of MMP10 in AW8507 (B) and CAL27 (F) cells. GAPDH was used for normalization. (C and G) Bar plot of Annexin V-FITC/PI staining indicating percentage of early-and late apoptotic cells in MMP10-knockdown and scrambled control clones of AW8507 (C) and CAL27 (G). (D and H) Representative scatterplots depicting PI (y-axis) vs. annexin V-FITC (x-axis) staining in MMP10 knockdown and scrambled control. In each scatterplot, top-left quadrant indicates necrotic cells, topright quadrant indicates late apoptotic cells, bottom-left quadrant indicates live cells and bottom-right quadrant
indicates early apoptotic cells. Data are shown as means ± SD. p-values are from Student's unpaired t-test and denoted as ns (not significant); ****, p < 0.0001. Data shown are representative of three independent experiments.
The protocol followed is mentioned in the materials and methods section as: "…Apoptosis assay Apoptosis was measured by double-labeling the cells with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide using FITC Annexin V Apoptosis Detection Kit, as per the manufacturer's instructions (Cat No. 556570, BD Pharmingen). Briefly, 0.3 million cells of AW8507-and CAL27-MMP10 knockdown along with scrambled control cells were re-suspended in 100 µl 1X Annexin V binding buffer and incubated on ice with 2 µl Annexin V-FITC conjugate for 15 min. This suspension was diluted to a final volume of 300 µl using ice-cold 1X Annexin V binding buffer and acquired immediately on AttuneNxT flow cytometer after addition of 1 µl Propidium iodide (PI). Data analysis was performed using FlowJo software. All the experiments including siRNA-mediated knockdown of MMP10 in both cell lines were performed three times.

Cell viability assay
Cell viability was assessed by resuspension of 0.3 million cells in 300 µl 1X Annexin V binding buffer and addition of 1 µl of 100 µg/ml Propidium iodide (PI). Percent viable and late apoptotic/necrotic cells were acquired on AttuneNxT flow cytometer. Data analysis was performed using FlowJo software. All the experiments were performed in three replicates…" Also, the data is explained in the results section as: "…To check if difference in proliferation rate is due to the difference in viability or proliferation or apoptosis. Using propidium iodide (PI) staining and flow cytometer-based sorting of live and dead cells, we conducted a cell viability study with shRNA-mediated MMP10 knockdown clones of AW8507 and CAL27. Results did not show significant difference in the viability of cells upon MMP10 knockdown (Supplementary Figure S3, A, E). The results were further supported by immunoblotting, which revealed comparable amounts of caspase-3 and PARP cleavage in the MMP10 knockdown clones of AW8507 and CAL27 compared to control cells (Supplementary Figure S3, A, E). We performed siRNAmediated knockdown of MMP10 in AW8507 and CAL27 cells. The knockdown of MMP10 was confirmed using real-time PCR (Supplementary Figure S3 Are the controls (values from technical repeats, individual experiments?) compared to all sh cells? The differences illustrated by these experiments seems clear, but statistics should be performed properly and described thoroughly. Without this information, it is impossible to evaluate the results. Response: We concur with the reviewer's comment. We have now clarified in the methods section and figure legends of the text how many times each experiment was conducted in order to allay the issue. All these experiments were performed in three replicates. As suggested by the reviewer, we have re-plotted all the invasion and wound healing experiment graphs and determined the p-values with the actual experimental values in Figure 2 (Figure 2, B-C, F-G, J-K) using unpaired student's ttest. Also, along with Figure 2, we have now re-plotted all the migration and invasion assay graphs in Figure 3, E-F, Figure 4, F-K, of the revised manuscript using actual experimental values. Earlier, we used relative values to control (=1 or 100) experimental data for plotting the graphs and deriving the p-values. For the cell proliferation assay ( Figure 2E, 2I), the p-values were determined by contrasting the control (sh-NT) with each of the three sh-MMP10 clones (sh-1, sh-2, and sh-3), while keeping the p-values determined using the actual experimental values or relative values to the control constant. We have now plotted the relative values to control after taking into account the reviewer's comment. We calculated and noted the p-values for each sh-MMP10 clone (sh-1, sh-2, and sh-3) compared to sh-NT control in the revised manuscript using Day 6/Day 1 ratio values.
Comment #12: Do the authors have reason to use P values <0.005 for ** and <0.0005 for *** as more commonly used values would be <0.01 and <0.001. Response: We agree with the reviewer's comments. The p-value range for *, ** and *** has been corrected in the manuscript figures and confirmed the same in the statistical analyses using GraphPad prism 8. The corrected range for p-values mentioned in the manuscript are as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Comment #14: How many times the blots were performed? For at least most important differences claimed based on the results, three blots should be performed and the differences in band intensities measured and related to housekeeping protein.

Response:
We agree with the reviewer. Three independent replicates of each Western blot were performed, in all our experiments. Using ImageJ software, we have now quantified the band intensities and compared them to the reference control. In the Supplementary Figures S14-S25, the quantified band intensities are shown as values underneath the original whole blot photographs. Figure 2, two separate blots for vinculin as housekeeping are shown. However, twelve proteins were detected and presumably, they are not all from two different blots with vinculin in addition. If this is the case, housekeeping should be provided for all blots separately. Without this information, it is impossible to evaluate the results. Response: In response to a reviewer's recommendation, we have individually added housekeeping genes for the blots in the updated Figure 4, A-B of the revised manuscript.

Comment #15: In
Comment #16: IHC: the authors do not provide information for the antibody used? Should there be also isotype control to show the antibody specificity -that depends on the antibody used. Without this information, it is impossible to evaluate the results. Response: We agree with the reviewer. The information on the MMP10 antibody (Human MMP10 Monoclonal Antibody (Cat No. MAB910, Bi Biotech)) utilised for the IHC experiment has been updated in the materials and methods section of the revised manuscript as "…The slides were blocked by horse serum for 1 h before incubating with the MMP10 primary antibody (Human MMP10 monoclonal antibody (Cat No. MAB910, Bi Biotech)) overnight at 4 o C in a moist chamber…". To demonstrate the antibody specificity, we included primary antibody-only and secondary antibody-only controls in the experiment (Supplementary Figure S1, A-B), as shown below in the figure in Comment #17 of Reviewer #1.

Comment #17:
The authors should describe in the text (and for this, please provide higher magnification images along with the current ones) where is the MMP10 IHC staining localized -is it inside the cells, on membrane, extracellular? Also, the distribution of MMP10 in tumors would be highly informative and interesting -e.g. is it expressed in the invasive front, close to certain tissue structures etc.? Response: Using IHC, we detected MMP10 staining in the cytoplasm of tumour cells. Compared to the centre of the tumour, the invasive front of tumours had higher expression levels of MMP10. As suggested by the reviewer, we have added the IHC localization data as Figure 1D, in the revised manuscript (as shown in the figure below). Accordingly, we have updated the results section of the manuscript as "…MMP10 protein was mainly detected in the cytoplasm of tumor cells ( Figure 1D). IHC results suggest significant overexpression of MMP10 protein in tongue cancer patients with lymph node metastasis (p=0.0075) ( Figure 1A, 1C, and Supplementary Figure S1C). Additionally, we find increased expression of MMP10 at the invasive fronts of the tumor compared to the centre of the tumor ( Figure 1D)…"

Reviewer #2
Comment #1: In Figure 1, authors showed that MMP10 overexpression was involved in tongue cancer patients with lymph node metastasis. However, authors previously reported that MMP10 overexpression was associated with lymph node metastasis in early tongue cancer patients. Even if the tumor size is small (T1 and T2), if patients have metastasis, authors cannot say early stage. Authors should not use the term "early stage". Moreover, authors should explain why you focused on T1 and T2 cancer. Moreover, in your previous study, authors used HPV negative early-stage tongue cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco. In general, cancer patients habitual of chewing betel nuts, areca nuts, lime or tobacco have buccal cancer. Why authors focused on tongue cancer in previous and current studies? Response: Based on the AJCC (American Joint Committee on Cancer)/ UICC (Union for International Cancer Control) TNM classification system (8th edition) guidelines, primary oral cavity tumors are clinically staged as T1 (measuring ≤2 cm) and T2 (measuring >2 cm but <4 cm), and are classified as early-stage. The occult lymph node metastasis present in these patients is not detected by physical examination or imaging techniques like PET/CT or ultrasonography, hence, these patients are termed as clinically early-stage oral cancer patients. As suggested by the reviewer, we have now included AJCC/UICC guidelines in the materials and methods section of the revised manuscript as "…Primary tongue tumors were staged as T1 (measuring ≤2 cm) or T2 (measuring >2 cm but <4 cm) as per AJCC (American Joint Committee on Cancer)/ UICC (Union for International Cancer Control) TNM classification system (8th edition) and referred as clinically early-stage tongue tumors…" We focused on T1 and T2 stage of tongue cancer since these individuals are known to have occult lymph node metastasis, which reduces their chance of survival by 50% when compared to those without lymph node metastases. We concur with the reviewer that individuals who regularly chew betel, areca, lime, or tobacco have an increased risk of developing tongue, buccal, and other oral cavity subsites cancer. It's interesting to note that tongue cancer is the most prevalent subtype in industrialised nations and is known to affect younger people more frequently in India (Sherin N. et al., Indian J. Cancer, 2008). Thus, the subject of our research was tongue cancer. 60% of patients with tongue cancer in our study chew tobacco, which is consistent with the literature and the reviewer's comments, as mentioned in the introduction. In addition, authors should check the phenotype of AXL overexpression. Response: In response to the reviewer's suggestions, we performed additional in vitro cell-based experiments and overexpressed AXL in AW1516 and the MMP10 knockdown clone (sh-2) of AW8507. The revised manuscript includes the assay results (Figure 4, A-K), which are described in the results section as: "…To study if AXL regulates MMP10-mediated proliferation, migration, and invasion of cells in the downstream, AXL overexpression and knockdown clones were used for in vitro metastasisrelated cell-based assays. AXL knockdown significantly reduced the biochemical levels of p-AKT, p-MTOR and p-NFKB levels and impeded the MMP10-driven proliferation, migration, and invasion phenotypes, whereas AXL overexpression clones restored the p-AKT and p-NFKB levels along with proliferation, migration, and invasion phenotypes of MMP10-knocked down AW8507 cells (Figure 4, A-K). These findings suggest that AXL is downstream of MMP10 and mediates the metastatic properties of tongue cancer cell lines upon upregulation by MMP10…"

Figure 4: miRNA-944/MMP10 induced EMT is mediated by the AXL signalling pathway. (A-B) Immunoblot of MMP10, p-AXL, AXL, p-AKT, AKT, p-mTOR, mTOR, p-NF-κB, NF-κB, Vinculin and β-actin upon overexpression/ knockdown of MMP10 or overexpression/ knockdown of AXL in tongue cancer cell lines. Vinculin or β-actin was used as loading control. Four Western blots probed for Vinculin are labelled with "v" symbol on the blot in the panels. For all other immunoblots in the panel, β-actin was used as loading control. (C-E) Cell proliferation assay of AW13516 cells stably overexpressing empty vector or AXL (C), AW8507-MMP10 KD cells stably overexpressing empty vector or AXL (D) and AW13516-MMP10 OE cells with scrambled siRNA (si-scr) or siRNA-mediated knockdown of AXL (E). Scatter plots indicate the total number of live cells on the mentioned day. (F, H and J) Boyden chamber migration and (G, I and K) invasion assays of AW13516 cells stably overexpressing empty vector or AXL (F and G), AW8507-MMP10 KD cells stably overexpressing empty vector or AXL (H and I) and AW13516-MMP10 OE cells with scrambled siRNA (si-scr) or siRNA-mediated knockdown of AXL (J and K) plotted as bar plots indicating the percentage of cells migrated or invaded, respectively. Representative images of crystal violet stained Boyden chambers. Data are shown as means ± SD. p-values are from Student's unpaired t-test and
denoted as ns (not significant); *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data shown are representative of three independent experiments. Comment #4: Authors showed that MMP10 overexpression promotes proliferation, migration, and invasion via AXL overexpression. How about the mechanism (how AXL regulates proliferation, migration, and invasion)? Response: We thank the reviewer for this pertinent query. To examine the activation of AXL pathway genes (p-AKT, p-mTOR, and p-NF-kB) affecting proliferation, migration, and invasion in tongue cancer cell lines, we generated additional reagents and performed experiments using these AXL overexpressing and knockdown clones. The results of the western blotting used to check for the expression of these AXL downstream genes are described in the results section and are included in the figures ( Figure 4B, as shown above in response to Comment #3 of the Reviewer #2) as: "…we investigated whether AXL overexpression in MMP10 knockdown clones or AXL knockdown in MMP10 overexpression clones would reverse the activation or inactivation of AXL downstream signaling, respectively. We performed stable overexpression of AXL in AW13516 (cell line with endogenously low levels of MMP10) and AW8507-MMP10 knockdown cells, and siRNA-mediated knockdown of AXL in AW13516-MMP10 overexpression clones. We found that overexpressing AXL activates AXL signalling in MMP10 knockdown cells whereas knocking down AXL inactivates AXL signalling in MMP10 overexpressing cells (Figure 4B), indicating that AXL signalling is downstream of MMP10…"

Reviewer #3
Comment #1: The main finding in the paper relies on the proposed regulatory mechanism of miR-944/MMP10/Axl axis. In this regard, it is not clear why the authors did not evaluate the basal expression level of Axl in all cell lines employed in this study. For instance, only the levels of MMP10 were demonstrated ( Figure S2). However, the authors did not show the levels of Axl in the cell. Does Axl levels correlate with MMP10 levels in all cell lines? And in tissue samples? Response: Following the reviewer's comments, we performed real-time PCR and Western analysis to screen for the expression of AXL transcript and protein in the tongue cancer cell lines (AW13516, AW8507 and CAL27). The data has been included in the Supplementary Figure S10, A-B, of the revised manuscript (as shown below). We had also screened for the expression of AXL transcript in tongue tumor samples (N=52) (Supplementary Figure S8 in the revised manuscript). Although the expression of AXL does not correlate with MMP10 expression in the cell lines, possibly due to limited number of cell lines, it shows significant correlation (p < 0.01) in tissue samples.

Supplementary Figure S10: Expression of AXL transcript and protein in tongue cancer cell lines. (A) qRT-PCR analysis for estimation of AXL in tongue cancer cell lines. Expression of AXL was normalized with GAPDH. Data are shown as means ± SD. (B) Immunoblot showing expression of p-AXL, AXL and β-actin protein in tongue cancer cell lines. Data shown are representative of three independent experiments.
Comment #2: What are the activation status (phosphorylated form) of Axl in the cell lines? And in the tissue samples? Response: Following the reviewer's query, we have performed Western blotting to screen for the expression of phosphorylated AXL in the TSCC cell lines (AW13516, AW8507 and CAL27). The data is included in the Supplementary Figure S10 of revised manuscript (as shown above in response to Comment #1 of the Reviewer #3). Screening for phosphorylated AXL in tissue samples would have been highly informative, however, we could not assess the expression due to limiting tissue samples.

Comment #3:
The mechanistic analysis was performed in MMP10 overexpressed cells. Considering that AW8507 and CAL27 express high levels of MMP10, why the authors overexpress this protein in a cell line and did not evaluate the mechanism in AW8507 and/or CAL27? Response: By knocking down MMP10, we have assessed the mechanistic analysis in the AW8507 and CAL27 cell lines. When MMP10 is silenced, we see the deactivation of the AXL signalling pathway. It's interesting to note that the AXL signalling pathway was reactivated when we overexpressed AXL in the MMP10 knockdown clones of AW8507 (sh-2). The results are further explained and covered in response to the comment #4 of the Reviewer #3 (below).
Comment #4: Since the authors are claiming that MMP10 induces Axl expression, it would be important to demonstrate whether MMP10 indeed modulates Axl and proteins in the Axl pathway expression. In this regard, would be interesting to use MMP10 knocked down and evaluate Axl and Axl pathway protein levels Response: We agree with the reviewer. In response to the query, we have evaluated AXL and AXL pathway proteins in the MMP10 knockdown clones of AW8507 and CAL27 clones. The western blotting results are included in Figure 4A (as shown above in response to Comment #3 of the Reviewer #2). We have detailed the experiment in the results section as "…we screened for the expression of AXL downstream signaling molecules (AKT, mTOR and NF-kB) upon overexpression and knockdown of MMP10. Results suggest that overexpression of MMP10 upregulates AXL and activates downstream signaling pathway whereas stable knockdown of MMP10 suppresses these effects ( Figure 4A)…" Comment #5: There is lack of consistency in the western blots. MMP10 expression levels are completely different for cell line AW13516 in figure 3I and figure S2C (Levels of MMP10 in figure S2 is different when compared to Figure 3I). Why did the authors used both GAPDH (figure S2C) and vinculin (figure 3I) as loading control? Response: We thank the reviewer for giving us an opportunity to clarify. Due to a discrepancy in exposure duration during blot creation in ChemiDoc, the MMP10 protein levels for the AW13516 cell line as shown in the western blotting data of Figure 3I (updated as Figure 4A in the revised manuscript) and Figure S2C were different. Additionally, there were differences in the amounts of protein loaded in the two gels (40 µg was loaded in the Western blot of Figure 3I versus 50 µg in Figure S2C). For better representation, we've replaced the western blot in Figure 3I (updated Figure 4A) with a comparable exposure blot loaded with the same quantity (50 µg) of protein.

Reviewer #4
Comment #1: Please consider revising the statement on the second sentence (rows 37-38). This result is not exactly clear in results section (see comment x) nor does the given n correspond to the number of node-positive tumors. Response: Our study includes a total of 219 early-stage tongue tumor samples. 81 samples were taken from patients positive for lymph node metastases of 219 samples. MMP10 is overexpressed in 70 of these 81 samples, or 86% of them. In response of the reviewer's comment, for clarity, the statement in abstract has been changed to read as "…Our immunohistochemical and real-time PCR analysis of 219 early-stage tongue tumors show MMP10 overexpression in 86% of node-positive tongue tumors (n=81; p<0.0001)…".

Comment #2:
The sentence starting from row 41 "Next, using orthotopic tongue cancer model…" gives the impression that all functional aspects (proliferation, migration and invasion) were studied in the mouse model, which based on the results, is not the case (they were studied in vitro). Please consider revising this sentence. Response: We thank the reviewers for the comment. The text in the abstract is re-written as "...We demonstrate that tongue cancer cell proliferation, migration, and invasion are suppressed by MMP10 knockdown or miR-944 overexpression. Next, we show that depletion of MMP10 prevents nodal metastases using an orthotopic tongue cancer mice model… " Comment #3: For Keywords, please consider including AXL or miRNAs instead of "Epithelial mesenchymal transition" as they could better reflect the results and conclusions of this manuscript. Response: We have updated the list of keywords with "AXL" and "miR-944" instead of "Epithelial mesenchymal transition". Comment #4: LNM is indeed found to result in worse prognosis for OTSCC patients, as suggested by authors in first part of the introduction (rows 60-62). Yet the references used do not discuss OTSCC but other cancers, although such research and associated literature exists. Please consider referring to OTSCC-related publications or modifying the sentence accordingly. Response: We apologize for the overlook on our part. We have updated following references indicating poor prognosis of OTSCC patients with lymph node metastasis in the introduction section of the revised manuscript. Comment #5: Row 70: Please consider the term "lymphogenesis", should it read "lymphangiogenesis" instead? Response: We thank the reviewer for the correction. We have now corrected the typographical error from "lymphogenesis" to "lymphangiogenesis".

Comment #6:
The sentence on rows 74-76 only refers to ovarian cancer, although he referenced studies also include HNSCC and cervical cancer. Please consider revising this sentence. Response: We have revised the sentence in the introduction section as "…Overexpression of MMP10 has been reported to promote invasion, metastasis and regulate stemness of cancer cells through activation of Wnt signaling pathway in head and neck, and ovarian cancer, and promote tumor progression by regulating angiogenic and apoptotic pathways in cervical cancer…" Comment #7: Please consider toning down the conclusion in the sentence on rows 79-81. Even if MMP10 have been upregulated in another head and neck cancer and relates to pre-metastatic niche formation in cancers overall, the variation between cancers overall and within HNSCCs is great. Thus, it is hard to convincingly suggest that MMP10 would play a LNM-related role in OTSCC based on these factors, although it warrants the research on this topic. Response: We agree and thank the reviewer for the suggestion. We have revised the text in the introduction as "…Additionally, MMP10 has been reported to promote pre-metastatic niche formation and found to be upregulated in early-stage esophageal cancer patients. However, the role of MMP10 remains unexplored in lymph node metastasis in early-stage tongue cancer…" Comment #8.1. This study focuses on tongue cancer, yet the patient cohorts also include tumors from the anatomical site of buccal mucosa. Could the authors explain why and describe, whether these tumors were excluded from the analyses or not? Response: Based on reviewer's suggestion, we performed additional analyses by excluding buccal mucosa samples--as presented in the graphs below (for reviewer's information only) -(A) graph showing the results of MMP10 immunohistochemistry, and (B) graph showing the delta CT values corresponding to the MMP10 transcript expression compared between node positive and node negative tongue tumor samples. These graphs are consistent with the graphs in Figures 1A and 1B (analysed by inclusion of buccal mucosa samples)--with no significant difference when buccal mucosa samples were included or excluded from the analyses. Moreover, the buccal mucosa patient samples were collected retrospectively from the N-zero clinical trial samples, we thus retained the buccal mucosa samples in the study.
A B Figure (A-B) The smoking status in our study refers to patients with habits of smoking tobacco as cigarettes, cigars or pipes whereas tobacco status represent the patients with the habit of smoke-less tobacco chewing. Based on reviewer's suggestion, we have included following text in the materials and methods section of the revised manuscript as: "…The patient details include age, gender, anatomic site, TNM tumor stage, nodal status, smoking (represents the patients with habits of smoking tobacco as cigarettes, cigars or pipes), chewing, alcohol, tobacco (represents the patients with the habit of smoke-less tobacco chewing), recurrence, metastasis and status at last follow-up…" Comment #8.3. What was the last follow-up date of these patients? Did it vary or were all followed to the suggested 5 years mark? Response: The last follow-up date of these patients ranges from 1 month to 11.7 years with a median follow-up of 3.9 years. As the patient samples were collected retrospectively, the follow-up period was not reached to 5-year mark in all patients. We have included this information in the materials and methods section of the revised manuscript as: "…status at last follow-up (the last follow-up date of patients ranges from 1 month to 11.7 years with a median follow-up of 3.9 years. As the patient samples were collected retrospectively, the follow-up period was not reached to 5-year mark in all patients)…" Comment #9: For western blotting (page 6 in M&M, page1-2 in supplementary M&M): How was the unspecific antibody binding blocked? Response: We have included the information about blocking step in materials and methods section as "…PVDF membrane blocking was performed using 5% BSA to avoid nonspecific binding of antibody…" Comment #10: In general, the methods describe both technical replicates in assays as well as number of repeated experiments in a varying manner resulting this information to be missing in some assays (e.g. boyden chamber assays). Could the authors please provide this information for all the assays when necessary (this suggestion includes supplementary M&M). Response: We have included the details regarding the number of times each experiment was repeated in the materials and methods section and figure legends (Figure 1, 2 Figure S2, S3, S7, S10) and mentioned the same in respective figure legends.

Figure 2, A, D, H: Genetic perturbation of MMP10 affects cell proliferation, migration, and invasion of tongue cancer cells. (A) qRT-PCR and immunoblot of MMP10 and GAPDH in AW13516 cells stably overexpressing empty vector or MMP10. Numbers on the blot indicate intensity ratio of MMP10 expression with respect to the vector control lane. (D and H) qRT-PCR and immunoblot of MMP10 and GAPDH in AW8507 (D) and CAL27 (H) cells with
sh-NT or stable MMP10 knockdown. Numbers on the blot indicate intensity ratio of MMP10 expression with respect to the sh-NT control lane.